Bacteria are often detected in the context of the disease they cause, but few infectious diseases are sufficiently specific that a physician can treat them directly. However, even for those diseases proof is necessary. The most common identification method is by direct culture of the specific pathogen, which is for many pathogens the “gold-standard” of detection and determination of the infectious disease. The second most used method for defining an infectious disease and its causative agent is indirect identification through antibody detection either to the whole bacterium, crude extracts, fractions of the pathogen, or single molecules. Indirect identification methods have the disadvantage of identifying other bacteria instead of the actual pathogen due to cross-reactivity of the extracts, fraction, or whole bacteria. Therefore, such tests only provide a first step for identification. Of particular interest are tests that focus on single molecules such as proteins. However, such tests have not been proven to be species-, subspecies-, or type-specific, and thus, share the same disadvantages as exist for whole extracts or fractions of whole bacteria (that is, cross-reactivity, as an example).
All bacteria consist of nucleic acids (RNA, DNA), proteins, saccharides, and lipids. Although these molecules may provide information for identifying bacterial species, subspecies and types, the most commonly used are nucleic acids and proteins. Comparative methods for chromosomal DNA and proteins have been developed to distinguish these species, but these molecules are not generally useful because of their low efficiency in specific techniques (DNA in PCR amplification, or cross-reactivity in hybridization techniques), or due to the large number of similar molecules within the proteome (several thousand molecules may have to be compared as a result of separation techniques having poor efficiency). There are fewer molecules belonging to the group of saccharides and lipids. However, methods for successful separation of saccharides are not yet available.
Despite these shortcomings, DNA sequences are presently used as amplification templates for identification of the bacterial species, subspecies, and types. Such DNA sequences are principally repetitive sequences for increasing the positive outcome of the PCR amplification procedure. Since these tests are multi-factorial, false-negative data that cannot be proven to be correct may result, and the tests do not have a “backup” target to verify a negative result.
The small subunit (SSU) rRNA has been found to be useful for distinguishing species from one another with a high degree of certainty. However, this marker can define an unknown strain of a species only if there is a certain taxonomical distance between this species and another. Classification/taxonomy defines the differences between bacteria. To determine a classification or taxonomic relationship, at least three bacteria are required, whereas to distinguish bacteria a minimum of two bacteria are required. Closely related species (for example, Mycobacterium avium and Mycobacterium intracellulare) cannot be distinguished with certainty by this method alone, and use of the SSU 16S rRNA sequence cannot distinguish bacterial subspecies and types since these subspecies have the same sequence. Furthermore, determining the SSU rRNA sequence requires multifactorial amplification by polymerase chain reaction.
Additional biological tests including DNA G+C content and chemotaxonomic methods, such as analysis of prominent cell wall molecules, may be required to obtain high confidence for species identification. Other investigations use the fatty acid composition to determine species. However, fatty acids must be generated by chemical reactions to release them from lipid molecules. Identification of bacterial subspecies and types may require other biochemical, enzymatic, and/or physiological methods, including information as to where those bacterial subspecies and types were obtained.
Species identification begins with isolation and growth of the bacterium either in-vitro or, if not possible, in-vivo, followed by extraction of chromosomal DNA and PCR amplification of the SSU 16S rRNA. However, in many situations the original sequence is not known and amplification processes cannot be performed. To reduce the number of possibilities, multiple standard biochemical, enzymatic, and physiological tests allow the determination of the bacterial species with high certainty for most bacterial species. However, to determine bacterial subspecies and types additional microbiological aspects must be considered, such as growth time, growth supplements, colony morphology, as examples.